InteractiveFly: GeneBrief

Hus1-like: Biological Overview | References

The checkpoint proteins Rad9, Rad1 and Hus1 form a clamp-like complex which plays a central role in the DNA-damage-induced checkpoint response. This study addresses the function of the 9-1-1 complex in Drosophila. This study analyzed the meiotic and somatic requirements of hus1. For that purpose, a null allele of hus1 was created by imprecise excision of a P element found 2 kb from the 3' of the hus1 gene. It was found that hus1 mutant flies are viable, but the females are sterile. hus1 mutant flies are sensitive to hydroxyurea and methyl methanesulfonate but not to X-rays, suggesting that hus1 is required for the activation of an S-phase checkpoint. It was also found that hus1 is not required for the G2-M checkpoint and for post-irradiation induction of apoptosis. Subsequently the role of hus1 in activation of the meiotic checkpoint was studied and it was found that the hus1 mutation suppresses the dorsal-ventral pattering defects caused by mutants in DNA repair enzymes. Interestingly, it was found that the hus1 mutant exhibits similar oocyte nuclear defects as those produced by mutations in DNA repair enzymes. These results demonstrate that hus1 is essential for the activation of the meiotic checkpoint and that hus1 is also required for the organization of the oocyte DNA, a function that might be independent of the meiotic checkpoint (Abdu, 2007).

In many cell types specific checkpoint mechanisms exist that monitor the integrity of the chromosomes. These checkpoints coordinate cell cycle progression with DNA repair to ensure the distribution of accurate copies of the genome to daughter cells. If left unrepaired, chromosomal lesions can lead to genomic instability, a major contributing factor in the development of cancer and other genetic diseases. The DNA damage checkpoint response system involves a signal transduction pathway consisting of sensors, transducers and effectors. Damaged DNA is initially sensed by a complex consisting of Hus1, Rad1 and Rad9 and the associated protein Rad17. Computer modeling suggests that Rad9, Hus1 and Rad1 (also called the 9-1-1 complex) form a doughnut-like heteromeric proliferating cell nuclear antigen (PCNA) complex that can be loaded directly onto damaged DNA (Rauen, 2000; Venclovas, 2000; Bermudez, 2003). The signal transducers comprise four sets of conserved protein families. One family is composed of ATM and ATM-Rad3-related (ATR) proteins. Downstream of these proteins are two sets of checkpoint kinases, the Chk1 and the Chk2 kinases and their homologues. The fourth conserved family is that of the BRCT-repeat-containing proteins. Finally, a diverse range of effector proteins execute the function of the DNA damage response, which can lead to cell cycle arrest, apoptosis or activation of the DNA repair machinery (Abdu, 2007).

A number of checkpoint proteins that were initially characterized in budding and fission yeast, have counterparts in Drosophila, Caenorhabditis elegans and mammals, demonstrating the conservation of these surveillance mechanisms. Several checkpoint proteins have been characterized in Drosophila, mainly the ATM and/or ATR and the Chk1 and/or Chk2 transducer family of proteins. An ATR homolog in Drosophila is encoded by mei-41. mei-41 is essential for the DNA damage checkpoint in larval imaginal discs and neuroblasts and for the DNA replication checkpoint in the embryo. mei-41 also has an essential role during early nuclear divisions in embryos. In addition, mei-41 plays important roles during meiosis, where it has been proposed to monitor double-strand-break repair during meiotic crossing over, to regulate the progression of prophase I, and to enforce the metaphase I delay observed at the end of oogenesis. Drosophila ATM and ATR orthologs are required for different functions. In Drosophila, recognition of chromosome ends by ATM prevents telomere fusion and apoptosis, by recruiting chromatin-modifying complexes to telomeres. It has also been shown that dATM and mei-41 have temporally distinct roles in G2 arrest after irradiation (Abdu, 2007 and references therein).

A Chk1 homolog in Drosophila is encoded by grapes. Similarly to mei-41, grapes is required to delay the entry into mitosis in larval imaginal discs after irradiation and to delay the entry into mitosis after incomplete DNA replication in the embryo. The Drosophila Chk2 homolog [also designated loki (lok) or Dmnk] regulates multiple DNA repair and apoptotic pathways following DNA damage. It plays an important role in a mitotic checkpoint in syncytial embryos and is important in centrosome inactivation. Like Mei-41, DmChk2 also plays an important role in monitoring double-strand-break repair during meiotic crossing over. Although understanding of the role of DNA damage proteins is increasing, there is still a lack of information on the function of the Drosophila PCNA-like complex, 9-1-1 (Abdu, 2007).

This study analyzes the interaction between the Drosophila Rad9, Hus1 and Rad1 proteins using a yeast two-hybrid assay. Interaction was detected between Hus1 and Rad9 or Rad1, but not between Rad9 and Rad1. This analysis focuses on the meiotic and somatic requirement of Hus1. A null allele of hus1 was created by imprecise excision of a P element. Sensitivity was observed of hus1 mutants to hydroxyurea (HU) and to methyl methanesulfonate (MMS) but not to X-ray irradiation. This implies that hus1 is required for the DNA replication checkpoint. The ability of a mutation in hus1 to suppress the eggshell polarity defects detected in mutants affecting double strand DNA repair enzymes demonstrates that it is required for the activation of the meiotic checkpoint that leads to a strong reduction in the translation of gurken mRNA. The similarity of the defects in the organization of the DNA in the oocyte nucleus between hus1 mutants and mutations in DNA repair enzymes suggest that hus1 may act upstream of the DNA repair machinery (Abdu, 2007).

Hus1 interacted with Rad1 or Rad9, however no interaction between Rad1 and Rad9 was observed. The yeast two hybrid system may not be sensitive enough to pick up the interaction, since possibly the interaction between these two proteins is more transient than the interaction between Hus1 and the other proteins. Similar results were seen in C. elegans (Hofmann, 2002) where these proteins interact in vivo (Abdu, 2007).

Several studies have investigated the role of hus1 during development. In mouse, Hus1 is an essential gene, since its inactivation results in mid-gestational embryonic lethality due to widespread apoptosis. Also, loss of Hus1 leads to an accumulation of genome damage (Weiss, 2000). Both fission and budding yeast that lack hus1 fail to arrest the cell cycle after DNA damage or blockage of DNA synthesis (Enoch, 1992; Weinert, 1994; Kostrub, 1998). In C. elegans, although hus1 is not absolutely required for embryonic survival, a significant fraction of hus1 embryos die during embryogenesis, probably because of genomic instability. Also, hus1 mutants fail to induce apoptosis and proliferation arrest following DNA damage and show increased sensitivity to DNA damage-induced lethality (Hofmann, 2002). The Drosophila hus1 is not an essential gene, although similarly to in C. elegans, the female mutants are sterile; this is probably due to the defects in the organization of the DNA within the oocyte nucleus (Abdu, 2007).

In order to test for a requirement for Drosophila hus1 in response to genotoxic stress, the survival rates of flies were studied after exposure to HU, MMS and IR during larval development; hus1 mutant flies were sensitive only to HU and MMS. This result suggests that hus1 is required for the activation of an S-phase checkpoint. It is possible that this requirement is due to a role of hus1 in Chk1 (Grapes) activation after genotoxic stresses that affect S phase. In yeast and mice, hus1 has been shown to be required for Chk1 activation after replicative stress (Bao, 2004; Weiss, 2003) In Drosophila, mutations affecting grapes and mei-41 fail to show a decrease in BrdU-staining after irradiation, indicating a defect in an S-phase checkpoint, and it would, therefore, seem likely that Hus1 signals to activate Grapes (Chk1) through Mei-41 during S phase. An increase in aneuploid nuclei in hus1 mutants after MMS treatment is consistent with a requirement for hus1 in the response to DNA damage caused during S phase as it has been suggested in budding yeast that spontaneous chromosome loss is primarily suppressed by functional S-phase checkpoints and not by G2-M checkpoints. Since the hus1 mutant still exhibits cell cycle arrest after irradiation, hus1 does not seem to be required for the G2-M checkpoint that is dependent on Mei-41. Rather, the data suggest that hus1 is only required for certain DNA damage situations, and not for the same spectrum as Mei-41 (Abdu, 2007).

Activation of a meiotic checkpoint, also known as the pachytene checkpoint, in response to the persistence of unrepaired DSBs appears to be a conserved regulatory feature common to yeast, worms, flies and vertebrates. However, a requirement for the 9-1-1 complex in activation of the meiotic checkpoint has only been demonstrated in budding yeast. It was found that mutations in the yeast Hus1 homologue, Mec3, and the Rad1 homologue, Ddc-1, abolish the pachytene checkpoint in budding yeast (Hong, 2000). In Drosophila, mutations in the spindle class of double-strand break (DSB) DNA repair enzymes, such as spn-A (RAD51), spn-B (XRCC3), spn-C (HEL308), spn-D (Rad51C) and okra (Dmrad54), affect dorsal-ventral patterning in Drosophila oogenesis and cause defects in the appearance of the oocyte nucleus. Interestingly, the defects in dorsal-ventral patterning and in the oocyte nucleus are dependent upon activation of a meiotic checkpoint (Ghabrial, 1999; Abdu, 2002; Staeva-Vieira, 2003). hus1 mutants are able to suppress the dorsal-ventral defects but not the defects in the organization of the DNA within the oocyte nucleus. The suppression of the DV patterning defects of spn-B mutants demonstrates that during meiosis Hus1 is required for the meiotic checkpoint in response to persistent DSBs. This finding is interesting in light of the fact that hus1 mutants are not IR sensitive or defective in somatic checkpoints after irradiation. Either there is a fundamental difference between germline and somatic DSBs and DSB response machinery, or the non-DSB lesions created during irradiation that are not present during meiotic recombination serve as triggers for an alternative sensing mechanism that does not require hus1 and is therefore still able to activate a checkpoint mechanism. The inability of hus1 mutants to suppress the karyosome phenotype along with the hus1 mutant phenotype by itself, demonstrates that hus1 is required for the organization of the oocyte DNA, a function that might be independent of the meiotic checkpoint (Abdu, 2007).

In this study it has been shown that Drosophila Hus1 is required for both the meiotic and somatic DNA damage responses as well as demonstrating a novel role of Hus1 in the organization of the oocyte nuclear DNA. Whereas some of the functions of Hus1, such as binding to 9-1-1 complex members and an essential role in surviving genotoxic stress during S phase, appear to be conserved across the species studied so far, some Hus1 functions seem to be less conserved. In contrast to the findings in yeast, worms and mouse, fly Hus1 is not required for survival after irradiation. Finally, the karyosome defect of hus1 mutants demonstrates a role for Drosophila Hus1 in organizing the chromosomal DNA of the meiotic nucleus (Abdu, 2007).

The Rad9-Hus1-Rad1 (9-1-1) clamp activates checkpoint signaling via TopBP1

DNA replication stress triggers the activation of Checkpoint Kinase 1 (Chk1) in a pathway that requires the independent chromatin loading of the ATRIP-ATR (ATR-interacting protein/ATM [ataxia-telangiectasia mutated]-Rad3-related kinase) complex and the Rad9-Hus1-Rad1 (9-1-1) clamp. Rad9ís role in Chk1 activation is to bind TopBP1, which stimulates ATR-mediated Chk1 phosphorylation via TopBP1ís activation domain (AD), a domain that binds and activates ATR. Notably, fusion of the AD to proliferating cell nuclear antigen (PCNA) or histone H2B bypasses the requirement for the 9-1-1 clamp, indicating that the 9-1-1 clampís primary role in activating Chk1 is to localize the AD to a stalled replication fork (Delacroix, 2007).

Genotoxic damage activates conserved checkpoint signaling pathways that maintain genomic stability by regulating cell cycle progression, triggering apoptosis, and influencing DNA repair. One pathway that is potently activated by replication stress leads to activation of Checkpoint Kinase 1 (Chk1), which promotes cell survival by blocking the firing of replication origins, preventing entry into mitosis, stabilizing stalled replication forks, and facilitating DNA repair. This pathway is initiated when the replicative DNA polymerases stall and large tracts of single-stranded DNA are created by the uncoupling of the replicative helicase from the advancing replication fork. The single-stranded DNA is then coated by replication protein A (RPA), which signals the independent recruitment of two checkpoint complexes: the ataxia-telangiectasia mutated (ATM)-Rad3-related kinase-ATR-interacting protein (ATR-ATRIP) complex and the Rad9-Hus1-Rad1 (9-1-1) complex. The ATRIP-ATR complex is bound to DNA by a direct interaction between ATRIP and RPA. In contrast, loading of the 9-1-1 complex requires several steps. First, DNA polymerase is recruited, which in turn recruits the clamp loader, Rad17-replication factor C (RFC) (You, 2002; Lee, 2003; Byun, 2005). Second, the Rad17-RFC then loads the proliferating cell nuclear antigen (PCNA)-like 9-1-1 complex onto chromatin in a reaction that is analogous to the loading of PCNA onto sites of DNA replication (Bermudez, 2003; Ellison, 2003). Although the binding of the ATRIP-ATR complex and the loading of the 9-1-1 complex occur independently of one another, both events are essential for optimal ATR-mediated Chk1 phosphorylation and activation (Melo, 2002; Delacroix, 2007 and references therein).

Despite the tremendous progress that has been made in deciphering the biochemical functions of the 9-1-1 complex and the in-depth understanding of the signals that lead to the loading of the 9-1-1 clamp, it has remained unclear how the chromatin-bound 9-1-1 complex initiates and propagates the Chk1-activating signal. Several studies have demonstrated that Rad9 orthologs in Schizosaccharomyces pombe, Saccharomyces cerevisiae, and humans interact with their respective TopBP1 orthologs (Cut4, Dpb11, and TopBP1). However, the significance of the Rad9-TopBP1 interaction in 9-1-1 function has not been explored. This study shows that the role of the 9-1-1 clamp is to recruit TopBP1, which then triggers ATR-mediated Chk1 phosphorylation. Thus, TopBP1 is a molecular bridge that links the independently recruited 9-1-1 and ATRIP-ATR complexes, leading to checkpoint activation (Delacroix, 2007).

Search PubMed for articles about Drosophila hus1

Abdu, U., Brodsky, M. and Schüpbach, T. (2002). Activation of a meiotic checkpoint during Drosophila oogenesis regulates the translation of Gurken through Chk2/Mnk. Curr. Biol. 12, 1645-1651. PubMed citation: 12361566

Abdu, U., Klovstad, M., Butin-Israeli, V., Bakhrat, A. and Schüpbach T. (2007). An essential role for Drosophila hus1 in somatic and meiotic DNA damage responses. J. Cell Sci. 120(Pt 6): 1042-9. PubMed citation: 17327271

Bao, S., Lu, T., Wang, X., Zheng, H., Wang, L. E., Wei, Q., Hittelman, W. N. and Li, L. (2004). Disruption of the Rad9/Rad1/Hus1 (9-1-1) complex leads to checkpoint signaling and replication defects. Oncogene 23: 5586-5593. PubMed citation: 15184880

Bermudez, V. P., Lindsey-Boltz, L. A., Cesare, A. J., Maniwa, Y., Griffith, J. D., Hurwitz, J. and Sancar, A. (2003). Loading of the human 9-1-1 checkpoint complex onto DNA by the checkpoint clamp loader hRad17-replication factor C complex in vitro. Proc. Natl. Acad. Sci. 100: 1633-1638. PubMed citation: 12578958

Byun, T. S., Pacek, M., Yee, M. C., Walter, J. C., and Cimprich, K. A. (2005). Functional uncoupling of MCM helicase and DNA polymerase activities activates the ATR-dependent checkpoint. Genes Dev. 19: 1040-1052. PubMed citation: 15833913

Delacroix, S., Wagner, J. M., Kobayashi, M., Yamamoto, K. and Karnitz, L. M. (2007). The Rad9-Hus1-Rad1 (9-1-1) clamp activates checkpoint signaling via TopBP1. Genes Dev. 21(12): 1472-7. PubMed citation: 17575048

Ellison, V. and Stillman, B. (2003). Biochemical characterization of DNA damage checkpoint complexes: Clamp loader and clamp complexes with specificity for 5' recessed DNA. PLoS Biol. 1: 231-243. PubMed citation: 14624239

Enoch, T., Carr, A. M. and Nurse, P. (1992). Fission yeast genes involved in coupling mitosis to completion of DNA replication. Genes Dev. 6: 2035-2046. PubMed citation: 1427071

Ghabrial, A. and Schüpbach, T. (1999). Activation of a meiotic checkpoint regulates translation of Gurken during Drosophila oogenesis. Nat. Cell Biol. 1: 354-357. PubMed citation: 10559962

Hofmann, E. R., Milstein, S., Boulton, S. J., Ye, M., Hofmann, J. J., Stergiou, L., Gartner, A., Vidal, M. and Hengartner, M. O. (2002). Caenorhabditis elegans HUS1 is a DNA damage checkpoint protein required for genome stability and EGL-1-mediated apoptosis. Curr. Biol. 12: 1908-1918. PubMed citation: 12445383

Hong, E. J. and Roeder, G. S. (2002). A role for Ddc1 in signaling meiotic double-strand breaks at the pachytene checkpoint. Genes Dev. 16: 363-376. PubMed citation: 12445383

Kostrub, C. F., Knudsen, K., Subramani, S. and Enoch, T. (1998). Hus1p, a conserved fission yeast checkpoint protein, interacts with Rad1p and is phosphorylated in response to DNA damage. EMBO J. 17: 2055-2066. PubMed citation: 9524127

Lee, J., Kumagai, A., and Dunphy, W. G. (2003). Claspin, a Chk1-regulatory protein, monitors DNA replication on chromatin independently of RPA, ATR, and Rad17. Mol. Cell 11: 329-340. PubMed citation: 12620222

Melo, J. and Toczyski, D. (2002). A unified view of the DNA-damage checkpoint. Curr. Opin. Cell Biol. 14: 237-245. PubMed citation: 11891124

Rauen, M., Burtelow, M. A., Dufault, V. M. and Karnitz, L. M. (2000). The human checkpoint protein hRad17 interacts with the PCNA-like proteins hRad1, hHus1, and hRad9. J. Biol. Chem. 275: 29767-29771. PubMed citation: 10884395

Staeva-Vieira, E., Yoo, S. and Lehmann, R. (2003). An essential role of DmRad51/SpnA in DNA repair and meiotic checkpoint control. EMBO J. 22: 5863-5874. PubMed citation: 14592983

Venclovas, C. and Thelen, M. P. (2000). Structure-based predictions of Rad1, Rad9, Hus1 and Rad17 participation in sliding clamp and clamp-loading complexes. Nucleic Acids Res. 28: 2481-2493. PubMed citation: 10871397

Weinert, T. A., Kiser, G. L. and Hartwell, L. H. (1994). Mitotic checkpoint genes in budding yeast and the dependence of mitosis on DNA replication and repair. Genes Dev. 18: 652-665. PubMed citation: 7926756

Weiss, R. S., Leder, P. and Vaziri, C. (2003). Critical role for mouse Hus1 in an S-phase DNA damage cell cycle checkpoint. Mol. Cell. Biol. 23: 791-803. PubMed citation: 12529385

You, Z., Kong, L., and Newport, J. (2002). The role of single-stranded DNA and polymerase in establishing the ATR, Hus1 DNA replication checkpoint. J. Biol. Chem. 277: 27088-27093. PubMed citation: 12015327

date revised: 27 June 2008

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